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Abstract:

Disclosed herein are compositions comprising cross-linkers for
cross-linking a retention vehicle polymer. The compositions are
particularly useful for local administration of a bioactive agent,
wherein prolonged or extended availability of the bioactive agent at the
site of administration is desired. Also disclosed are methods of
delivering the compositions to a subject.

Claims:

1. A composition comprising: a retention vehicle polymer; and a
controlled release cross-linking agent for cross-linking the retention
vehicle polymer in situ in a subject.

11. A method for delivering a bioactive agent to a subject, comprising:
injecting into the subject a composition comprising: a biodegradable
polymeric microparticle comprising a bioactive agent encapsulated
therein; a retention vehicle polymer; and a controlled release
cross-linking agent for cross-linking the retention vehicle polymer in
situ in the subject.controlled release cross-linking agent

21. A biodegradable polymeric microparticle comprising a ligand that
binds to a joint tissue.

Description:

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority from prior U.S.
Provisional Application No. 61/318,574, filed Mar. 29, 2010, the entire
contents of which are incorporated herein by reference.

BACKGROUND

[0002] Drug delivery vehicles and pharmaceutical compositions are often
used to locally deliver bioactive agents to particular locations within a
subject. Many vehicles and compositions, however, are difficult to retain
locally for extended periods of time. Some formulations can quickly clear
(diffuse out of, migrate out of, and/or be removed by one or more
active-transport or passive-transport mechanisms) from the site of
administration. Rapid clearance of these vehicles and compositions can
necessitate their frequent re-administration (re-dosing) in order to
provide an effective treatment over a desired duration of days or months.
Delivery vehicle and/or drug diffusion away from the site of
administration can also induce unwanted side effects, such as
inflammatory responses and systemic side effects.

SUMMARY

[0003] Disclosed herein are compositions comprising cross-linkers for
cross-linking a retention vehicle polymer in a subject, to improve local
retention of an active microparticle (i.e., a microparticle containing a
bioactive agent) at and/or near the site of administration. The disclosed
compositions and methods are particularly useful for local administration
of a bioactive agent, wherein prolonged or extended availability of the
bioactive agent at the site of administration is desired.

[0004] The disclosed compositions comprise: a retention vehicle polymer;
and a controlled release cross-linking agent for cross-linking the
retention vehicle polymer in situ in a subject, and optionally comprise a
biodegradable polymeric microparticle comprising a bioactive agent
encapsulated therein.

[0005] The disclosed methods comprise administering one or more of the
disclosed compositions to a subject.

[0006] Also disclosed are microparticles comprising ligands for adhering
to a particular tissue, such as a joint tissue, e.g., a knee tissue.

[0007] The advantages of the invention will be set forth in part in the
description which follows, and in part will be obvious from the
description, or may be learned by practice of the aspects described
below. The advantages described below will be realized and attained by
means of the elements and combinations particularly pointed out in the
appended claims. It is to be understood that both the foregoing general
description and the following detailed descriptions are exemplary and
explanatory only and are not restrictive.

DETAILED DESCRIPTION

[0008] Before the present compounds, compositions, composites, articles,
devices and/or methods are disclosed and described, it is to be
understood that the aspects described below are not limited to specific
compounds, compositions, composites, articles, devices, methods, or uses
as such may, of course, vary. It is also to be understood that the
terminology used herein is for the purpose of describing particular
aspects only and is not intended to be limiting.

[0009] In this specification and in the claims that follow, reference will
be made to a number of terms that shall be defined to have the following
meanings:

[0010] Throughout this specification, unless the context requires
otherwise, the word "comprise," or variations such as "comprises" or
"comprising," will be understood to imply the inclusion of a stated
integer or step or group of integers or steps but not the exclusion of
any other integer or step or group of integers or steps.

[0011] It must be noted that, as used in the specification and the
appended claims, the singular forms "a," "an" and "the" include plural
referents unless the context clearly dictates otherwise. Thus, for
example, reference to "a bioactive agent" includes mixtures of two or
more such agents, and the like.

[0012] "Optional" or "optionally" means that the subsequently described
event or circumstance can or cannot occur, and that the description
includes instances where the event or circumstance occurs and instances
where it does not.

[0013] Ranges may be expressed herein as from "about" one particular
value, and/or to "about" another particular value. When such a range is
expressed, another aspect includes from the one particular value and/or
to the other particular value. Similarly, when values are expressed as
approximations, by use of the antecedent "about," it will be understood
that the particular value forms another aspect. It will be further
understood that the endpoints of each of the ranges are significant both
in relation to the other endpoint, and independently of the other
endpoint.

[0014] Disclosed are compounds, compositions, and components that can be
used for, can be used in conjunction with, can be used in preparation
for, or are products of the disclosed methods and compositions. These and
other materials are disclosed herein, and it is understood that when
combinations, subsets, interactions, groups, etc. of these materials are
disclosed that while specific reference of each various individual and
collective combinations and permutation of these compounds may not be
explicitly disclosed, each is specifically contemplated and described
herein. For example, if a number of different polymers and agents are
disclosed and discussed, each and every combination and permutation of
the polymer and agent are specifically contemplated unless specifically
indicated to the contrary. Thus, if a class of molecules A, B, and C are
disclosed as well as a class of molecules D, E, and F and an example of a
combination molecule, A-D is disclosed, then even if each is not
individually recited, each is individually and collectively contemplated.
Thus, in this example, each of the combinations A-E, A-F, B-D, B-E, B-F,
C-D, C-E, and C-F are specifically contemplated and should be considered
disclosed from disclosure of A, B, and C; D, E, and F; and the example
combination A-D. Likewise, any subset or combination of these is also
specifically contemplated and disclosed. Thus, for example, the sub-group
of A-E, B-F, and C-E are specifically contemplated and should be
considered disclosed from disclosure of A, B, and C; D, E, and F; and the
example combination A-D. This concept applies to all aspects of this
disclosure including, but not limited to, steps in methods of making and
using the disclosed compositions. Thus, if there are a variety of
additional steps that can be performed it is understood that each of
these additional steps can be performed with any specific embodiment or
combination of embodiments of the disclosed methods, and that each such
combination is specifically contemplated and should be considered
disclosed.

[0015] A "bioactive agent" refers to an agent that has biological
activity. The biological agent can be used to treat, diagnose, cure,
mitigate, prevent (i.e., prophylactically), ameliorate, modulate, or have
an otherwise favorable effect on a disease, disorder, infection, and the
like. A "releasable bioactive agent" is one that can be released from a
disclosed composition. Bioactive agents also include those substances
which affect the structure or function of a subject, or a pro-drug, which
becomes bioactive or more bioactive after it has been placed in a
predetermined physiological environment.

[0016] A "cross-linker" refers to an agent that forms a physical or
chemical bond between polymer chains of the retention vehicle polymer,
either between different polymer chains or between different parts of the
same polymer chain. For cross-links made with physical bonds, the
cross-linker can dissociate and leave the polymer chain. A
"controlled-release cross-linker" is a cross-linker that can replenish
dissociated cross-linker and maintain cross-linking with the polymer
chain(s) over an extended period of time, e.g., releasing cross-linker
for a week, a month or longer, to maintain the structure of retention
vehicle polymer.

[0017] A "retention vehicle polymer" refers to a polymer that can be
cross-linked to form a polymer network that retains a releasable
bioactive agent, e.g., a microencapsulated bioactive agent."

[0018] In one embodiment, the composition of the invention includes a
cross-linking agent for cross-linking a retention vehicle polymer, which
can enable prolonged or extended retention of a bioactive agent, e.g., a
microencapsulated bioactive agent, at a particular site within a subject.
In another embodiment, the composition of the invention includes a
microparticle comprising a ligand that can bind or adhere to a tissue of
a subject and provide an extended release of a bioactive agent to a local
site of administration.

[0019] The controlled release cross-linking agent is capable of
cross-linking the retention vehicle polymer in situ within a subject for
a period of time ranging from 1 day to 3 months or more, for example 2
weeks, 3 weeks, 1 month, 1.5 months, 2 months, 3 months, or more. The
extended and controlled release cross-linking activity is attributed to a
number of factors, depending on the exact composition. For example, when
poorly soluble salts are used as the controlled release cross-linker, as
specified herein, the salts slowly dissolve at or near the site of
injection and continuously cross-link the retention vehicle polymer over
time. Additionally, extended cross-linking can cross-link endogenous
polymers, such as hyaluronic acid, which are typically present at or near
joints, such as the knee.

[0020] In some embodiments, the compositions of the invention include a
cross-linker that enables a retention vehicle or a fluid within a subject
to effectively retain a pharmaceutical formulation including a bioactive
agent, e.g., a microencapsulated bioactive agent, at a desired location
for an extended period of time, such as days or even months. The ability
to retain the pharmaceutical formulation at a local site for an extended
period of time allows for extended therapy, and may reduce the need for
re-dosing, as is commonly required in certain locally administered
therapies. Local retention of the pharmaceutical composition may also
reduce unwanted side effects that can occur when components of the
pharmaceutical formulation migrate or otherwise escape the targeted
therapeutic site.

[0021] The compositions of the invention are particularly useful for areas
within a subject, such as a human, that are in or near joints or in or
near the subarachnoid area of the brain. When locally administering
pharmaceutical compositions to these areas, bioactive agents and other
components of pharmaceutical formulations are particularly susceptible to
migration or escape from the local site. Examples of locations where the
compositions of the invention can provide improved retention include, but
are not limited to, the hip, knees, shoulders, ankles, elbows, wrists,
toes, fingers, and spinal facet joints, and areas in the brain such as
the subarachnoid area.

[0022] Joints, in particular, have opposing bones having respective
opposing hyaline cartilage articular surfaces, a peripheral, collagenous
ligamentous capsule connecting the articular surfaces and defining a
central joint space and a synovium lining upon an inner wall of the
capsule, and also include synovial fluid contained within the joint
space. Synovial fluid contained within the joint space naturally contains
polymers, such as hyaluronic acid, that helps to retain pharmaceutical
compositions within the joint. However, synovial fluid is frequently
regenerated. As synovial fluid regenerates, any pharmaceutical
composition including a bioactive agent is susceptible to migration away
from or completely out of the joint. Thus, synovial fluid, in of itself,
is not optimal for retaining a pharmaceutical composition. The
compositions of the invention can be useful in reducing synovial fluid
movement and therefore can improve the ability of synovial fluid to
retain a pharmaceutical composition or a bioactive agent.

[0023] The compositions disclosed herein can result in the local
retainment of a biodegradable microparticle containing a bioactive agent
(which can be released from the microparticle over time) for a period of
time ranging from 1 week to 3 months or longer, i.e., the retention
vehicle polymer, by virtue of the cross-linker, can retain the active
microparticle at or near the site of original administration for a period
of time ranging from 1 week to 3 months or longer. Depending on the
nature of the cross-linker and exact composition administered to the
subject, active microparticle retention times of 1 week, 2 weeks, 3
weeks, 1 month, 1.5 months, 2 months, 2.5 months, 3 months, or longer,
can be achieved. Endogenous retention vehicle polymers produced naturally
at or near the site of administration, such as hyaluronic acid naturally
produced in or near joints, such as the knee, can be cross-linked to
assist in the retention of the active microparticle at the site of
injection.

[0024] One embodiment of the invention includes a composition suitable for
administration to a subject that comprises a controlled release
cross-linking agent capable of cross-linking a polymer or higher order
polymeric structure, such as a microparticle, to thereby improve
retention of a bioactive agent or pharmaceutical composition within a
local target area of a subject. In one embodiment, the polymer is already
present in a subject and can be a naturally occurring polymer present in
the subject. An example is hyaluronic acid, which is a constituent of
synovial fluid. The compositions of the invention provide a controlled
release cross-linking agent that can cross-link the hyaluronic acid
present in synovial fluid and therefore reduce movement of the synovial
fluid or the hyaluronic acid. The bioactive agent or pharmaceutical
composition can therefore be retained in the synovial fluid for longer
periods of time.

[0025] The controlled release cross-linking agent can vary in composition
depending on the particular retention vehicle for which cross-linking is
desired. Generally, the controlled release cross-linking agent has at
least two reactive groups or is multivalent, such that the controlled
release cross-linking agent is capable cross-linking two or more polymer
chains together. The cross-linking can be achieved with physical bonds,
such as ionic and/or hydrogen bonds, or the cross-linking can be achieved
with chemical bonds. Examples of controlled release cross-linking agents
for polysaccharides, charged polymers, or polymers with polar functional
groups include a variety of inorganic salts that include multivalent
cations and/or anions. Examples include salts of calcium, zinc,
strontium, magnesium, barium, manganese, or other multivalent ions. Such
salts can comprise any suitable anion, such as chloride, among others.

[0026] Other salts that can be used include salts of transition metals,
such as Cu(II) and Fe(II). It was observed that such salts can cross-link
hyaluronic acids (HA) of varying molecular weights when present in
aqueous compositions including from 1-2% by weight hyaluronic acid and
from 1-2 mM metal salt (concentration relative to total composition
including aqueous component); see Table 1.

[0027] Specific compositions of the invention include those containing a
biodegradable polymeric microparticle (e.g., poly(lactide),
poly(glycolide), poly(caprolactone), or a combination or copolymer
thereof) comprising a bioactive agent, as specified herein, together with
one or more of the hyaluronic acid polymers and metal salts listed in
Table 1, in any suitable concentration, such as those listed in Table 1.
As discussed elsewhere, such compositions can be injected into a subject
to improve local retention of the microparticle containing the bioactive
agent, such as in or near a joint, such as the knee, or near certain
areas of the brain.

[0028] Other suitable controlled release cross-linking agents include
small molecules, biomolecules, polymers, or biopolymers with charged or
polar functional groups, such as peptides, proteins, and the like. The
controlled release cross-linking agent can, in some aspects, have
biological activity in of itself, and thus can be a bioactive agent. The
controlled release cross-linking agent can also be an inorganic charged
or multivalent particle, such as an inorganic microparticle or
nanoparticle that is pharmaceutically acceptable. The controlled release
cross-linking agent, in some aspects, can be a poorly soluble salt, such
as calcium chloride, zinc chloride, or magnesium chloride. The controlled
release cross-linking agent can be a bioresorbable microparticle or
nanoparticle with charge on its surface. Such cross-linking
microparticles or nanoparticles can have bioactive agent inside or no
bioactive agent inside. In other aspects, polyethylene glycol (PEG) can
be used as a controlled release cross-linking agent. For example, two PEG
end-groups can be used to cross-link two or more polymer chains together,
such as two or more polysaccharide (e.g., hyaluronic acid) polymer
chains.

[0029] In some embodiments, the compositions of the invention include the
retention vehicle and the controlled release cross-linking agent. The
composition including the controlled release cross-linking agent can also
be separately administered from another composition that includes the
retention vehicle. Suitable retention vehicles include a variety of
polymers, such as polyelectrolytes, and polysaccharides, such as
hyaluronic acid, alginate, chitosan, collagen, fucans, cellulose,
including methylcellulose, hydroxypropylcellulose,
hydroxypropylmethylcellulose, carboxymethylcellulose, cellulose acetate
phthalate, cellulose acetate succinate, hydroxypropylmethylcellulose
phthalate; casein, dextrans, and starches such as amylose and
amylopectin, among others. Polyethylene glycol (PEG) can also be used as
a retention polymer vehicle.

[0030] In one embodiment the retention vehicle is hyaluronic acid (also
known as hyaluronan, hyaluronate, and HA), which is an anionic,
non-sulfated glycosaminoglycan distributed widely throughout connective,
epithelial, and neural tissues. The hyaluronic acid present in the
composition can be the same or different than hyaluronic acid that is
naturally present in synovial fluid. Polysaccharides such as hyaluronic
acid are capable of being cross-linked through a variety of mechanisms,
which enhances the viscosity of the formulation and also helps to improve
retention in a location of subject. Hyaluronic acid polymers can also
function as tissue adhesives such that when the polymer is injected into
a tissue, muscle diffusion and migration of the polymer through fascial
planes in minimized. See e.g. Cohen et al. Biophys J. 2003; 85:
1996-2005. The tissue adhesion and therefore low tissue migration
characteristic of a formulation which comprises hyaluronic acid therefore
enables the formulation to remain largely at the administration or
injection site. The hyaluronic acid formulations of the invention can
therefore limit drug or biologic exposure to surrounding or adjacent
non-target tissues, thereby reducing side effects.

[0032] The retention vehicle can be cross-linked in the composition, prior
to administration to the subject. The retention vehicle can also be
non-cross-linked in a first composition and can encounter the
cross-linker in the subject and/or can be co-administered with a separate
composition that comprises the controlled release cross-linking agent.

[0033] Various degrees of retention vehicle cross-linking can be used,
e.g., light cross-linking (10% or less) to heavy cross-linking (80% or
more). Generally, cross-linking density can be controlled by molecular
weight of the retention vehicle polymer (e.g., lower molecular weight
polymers can be used to get achieve higher cross-linking density),
concentration of the cross-linker, concentration of the polymer, type of
cross-linker, or by using one or more cross-linkers, or a combination
thereof.

[0034] In some embodiments, the retention vehicle can include two or more
different polymers that are complexed or cross-linked together. For
example, hyaluronic acid polymers can be cross-linked with other
polymers, such as alginate. To make such a cross-linked polymer, alginate
and hyaluronic acid can be admixed to form a homogeneous solution prior
to initiating the cross-linking reaction. Calcium ions, for example, can
be added to the reaction mixture, e.g., in the form of a calcium chloride
solution, resulting in the formation of ionic cross-links between
hyaluronic acid and alginate. Hyaluronic acid and alginate can also be
cross-linked via both hydroxyl and carboxyl groups, resulting in ether
and ester cross-linking bonds. Other anionic polymers, such as
carboxymethylcellulose and gellan gum, and others, can be cross-linked
with hyaluronic acid in a similar manner.

[0035] In some embodiments, the cross-linker is present in a delivery
vehicle that enables controlled or extended release of the controlled
release cross-linking agent over time. The delivery vehicle can provide
an extended dose of the controlled release cross-linking agent for
continuously cross-linking the retention vehicle. For example, a delivery
vehicle comprising the controlled release cross-linking agent can be
administered along with or separate from a bioactive agent to a joint.
The delivery vehicle can provide an extended supply of controlled release
cross-linking agent to synovial fluid, such that as the synovial fluid
regenerates, fresh synovial fluid can be cross-linked and therefore more
effectively retain the bioactive agent or pharmaceutical composition. A
similar effect can be achieved with an exogenous retention vehicle that
is administered to the subject.

[0036] An exemplary formulation for injection comprises a 2% or higher
solution of hyaluronic acid (e.g., 2% or higher, 3% or higher, 4% or
higher, including 5%, 10%, and 20%) and controlled release cross-linking
agent (in any concentration) in an aqueous composition or
phosphate-buffered saline (PBS). Such an exemplary formulation can be
used to suspend and inject microparticles containing a bioactive agent
with good resistance to dispersion in water after the injection. This
allows for the active microspheres to stay localized at the site of
injection for open surgical procedures, for example. Such formulations
can be easily administered at a local site through a small gauge, or
large bore, needle, depending on the exact viscosity of the formulation.
Alternatively, such a formulation can be administered through a syringe
itself (without a needle) at an open site, or a site at which surgery is
being carried out, e.g., the tissue of a joint such as a knee during a
surgical knee operation, such as knee replacement surgery.

[0037] In general, a variety of delivery vehicles can be suitable for
providing a controlled or extended release of the controlled release
cross-linking agent to the local site of administration. These include
implant devices, implantable fibers, rods, viscous pharmaceutical
formulations, degradable pharmaceutical carriers, microparticles,
nanoparticles, ion-exchange polymers, particles of insoluble salts, and
the like.

[0038] A preferred delivery vehicle is a biodegradable polymeric
microparticle. The controlled release cross-linking agent can be present
within or on the microparticle. Two or microparticles can themselves be
cross-linked together with a substance that provides the controlled
release cross-linking agent as the substance degrades or dissociates, or
as the microparticles dissociate from each other. In one example, the
microparticle can be associated with or comprise a polymer that is
cross-linked with the controlled release cross-linking agent, which can
in some aspects, be the retention vehicle polymer. For example, the
microparticle can comprise a polysaccharide cross-linked with the
controlled release cross-linking agent, such as a hyaluronic acid
cross-linked with calcium chloride. Once administered, such a
polysaccharide can slowly dissociate to release the controlled release
cross-linking agent over time. Such a microparticle can be a nanoparticle
or a macroparticle. Furthermore, such particles can also be hydrogels.

[0039] Depending on the composition or the mode of administration, the
microparticle can comprise the controlled release cross-linking agent
and/or the bioactive agent and can in some aspects be present together
with the retention polymer. In one embodiment, the composition includes
the microparticle comprising the controlled release cross-linking agent.
Another composition can be separately administered which includes another
microparticle, which can be the same or different, comprising the
bioactive agent. Either of these compositions can be administered along
with the retention polymer, or can be administered directly into a local
site, without the retention polymer or separately from the retention
vehicle administration. For example, these compositions can be
administered into a joint that contains synovial fluid. The
microparticles can also be associated with, or cross-linked with, the
retention vehicle polymer. For example, the microparticles can be
cross-linked with a hyaluronic acid polymer. In some embodiments,
microparticles can be prepared from a suitable retention polymer, such as
a polysaccharide. These microparticles can be linked together with the
controlled release cross-linking agent, if desired. These microparticles
can also contain combinations of the retention polymer and any of the
biodegradable polymers disclosed below.

[0040] The microparticles generally range in size from 10 nm to 2000
microns. In some examples, the microparticles are from 1 to 80 microns in
diameter, from 5 to 60 microns in diameter, or from 10 to 50 microns in
diameter.

[0041] The microparticles preferably comprise a biodegradable polymer and
one or more of a bioactive agent and controlled release cross-linking
agent. Suitable biodegradable polymers for use with the invention include
without limitation poly(lactide), a poly(glycolide), a
poly(lactide-co-glycolide), a poly(caprolactone), a poly(orthoester), a
poly(phosphazene), a poly(hydroxybutyrate) a copolymer containing a
poly(hydroxybutarate), a poly(lactide-co-caprolactone), a polycarbonate,
a polyesteramide, a polyanhydride, a poly(dioxanone), a poly(alkylene
alkylate), a copolymer of polyethylene glycol and a polyorthoester, a
biodegradable polyurethane, a poly(amino acid), a polyamide, a
polyesteramide, a polyetherester, a polyacetal, a polycyanoacrylate, a
poly(oxyethylene)/poly(oxypropylene) copolymer, polyacetals, polyketals,
polyphosphoesters, polyhydroxyvalerates or a copolymer containing a
polyhydroxyvalerate, polyalkylene oxalates, polyalkylene succinates,
poly(maleic acid), and copolymers, terpolymers, combinations thereof.

[0042] The biodegradable polymer can comprise one or more residues of
lactic acid, glycolic acid, lactide, glycolide, caprolactone,
hydroxybutyrate, hydroxyvalerates, dioxanones, polyethylene glycol (PEG),
polyethylene oxide, or a combination thereof. More preferably, the
hydrophobic polysaccharide derivative is blended with one or more
polymers that comprise one or more residues of lactide, glycolide,
caprolactone, or a combination thereof.

[0043] In some aspects, the biodegradable polymer comprises one or more
lactide residues. The polymer can comprise any lactide residue, including
all racemic and stereospecific forms of lactide, including, but not
limited to, L-lactide, D-lactide, and D,L-lactide, or a mixture thereof.
Useful polymers comprising lactide include, but are not limited to
poly(L-lactide), poly(D-lactide), and poly(DL-lactide); and
poly(lactide-co-glycolide), including poly(L-lactide-co-glycolide),
poly(D-lactide-co-glycolide), and poly(DL-lactide-co-glycolide); or
copolymers, terpolymers, combinations, or blends thereof.
Lactide/glycolide polymers can be conveniently made by melt
polymerization through ring opening of lactide and glycolide monomers.
Additionally, racemic DL-lactide, L-lactide, and D-lactide polymers are
commercially available. The L-polymers are more crystalline and resorb
slower than DL-polymers. In addition to copolymers comprising glycolide
and DL-lactide or L-lactide, copolymers of L-lactide and DL-lactide are
commercially available. Homopolymers of lactide or glycolide are also
commercially available.

[0044] When poly(lactide-co-glycolide), poly(lactide), or poly(glycolide)
is used, the amount of lactide and glycolide in the polymer can vary. For
example, the biodegradable polymer can contain 0 to 100 mole %, 40 to 100
mole %, 50 to 100 mole %, 60 to 100 mole %, 70 to 100 mole %, or 80 to
100 mole % lactide and from 0 to 100 mole %, 0 to 60 mole %, 10 to 40
mole %, 20 to 40 mole %, or 30 to 40 mole % glycolide, wherein the amount
of lactide and glycolide is 100 mole %. In a further aspect, the
biodegradable polymer can be poly(lactide), 95:5
poly(lactide-co-glycolide) 85:15 poly(lactide-co-glycolide), 75:25
poly(lactide-co-glycolide), 65:35 poly(lactide-co-glycolide), or 50:50
poly(lactide-co-glycolide), where the ratios are mole ratios.

[0045] In a further aspect, the biodegradable polymer can comprise a
poly(caprolactone) or a poly(lactide-co-caprolactone). For example, the
polymer can be a poly(lactide-caprolactone), which, in various aspects,
can be 95:5 poly(lactide-co-caprolactone), 85:15
poly(lactide-co-caprolactone), 75:25 poly(lactide-co-caprolactone), 65:35
poly(lactide-co-caprolactone), or 50:50 poly(lactide-co-caprolactone),
where the ratios are mole ratios.

[0046] In a separate embodiment of the invention, a composition comprises
a microparticle or nanoparticle comprising a ligand that can bind to a
particular tissue, bone or cartilage to enhance retention of the
microparticle in a particular location of a subject. For example, a
ligand can be conjugated to the surface of the microparticles such that
the ligand or targeting moiety binds the microparticles to a specific
target, epitope or receptor site on tissue, bone or cartilage. For
example, a targeting moiety (e.g. antibody or fragment) can be attached
to the polymer directly or via a linker or via a biosensitive linker. The
targeting moiety can act to deliver or localize the polymer microparticle
to a particular area of a subject. Non-limiting examples of targeting
agents or moieties can include folate-binding agents, biotin, albumin,
peptides, proteins, polysaccharides, RGD peptides, glycosylated targeting
ligands, lipoproteins, antibodies, antibody fragments, enzymes, nucleic
acids, aptamers, tumor-specific ligands or peptides, receptor-specific
ligands or peptides, among others. Surface functionalization can be
accomplished by covalently linking a secondary component to the
microparticle.

[0047] Covalent linking of a ligand can be achieved by a 3+2 cycloaddition
reaction between a reactive moiety on the polymeric microparticle and on
the secondary component. For example, the polymeric microparticle can
contain a diene moiety and the secondary component can contain a
dieneophile. Alternatively, the polymeric microparticle can contain a
dieneophile and the secondary component can contain a diene. Covalent
linking can be achieved by a 2+2 cycloaddition reaction between a
reactive moiety on the polymeric microparticle and on the secondary
component.

[0048] Covalent linking of a ligand can also involve linking a secondary
component to the microparticles through an ether, imidate, thioimidate,.
ester, amide, thioether, thioester, thioamide, carbamate, disulfide,
hydrazide, hydrazone, oxime ether, oxime ester, and/or 30 amine linkage.
Such linkages can be formed from known covalent coupling chemistries as
amine-reactive chemistries, thiol-reactive chemistries,
carboxylate-reactive chemistries, hydroxyl-reactive chemistries,
aldehyde, and ketone-reactive chemistries, active hydrogen-reactive
chemistries, photoreactive chemical reactions, redox-based chemistries,
and the like. In one example, if the secondary component or the polymer
particle has an amino group and the other has a carboxylate group, they
can be covalently linked via a peptide bond. This can typically be
accomplished by using an activating agent to mediate the coupling.
Various activating agents that can be used for the coupling reaction
include, but are not limited to,
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC),
dicyclohexylcarbodiimide (DCC), N,N-diisopropyl-carbodiimide (DIP),
benzotriazol-lyl-oxy-tris-(dimethyl amino)phosphonium
hexa-fluorophosphate (BOP), hydroxybenzotriazole (HOBt), and
N-methylmorpholine (NMM), including a mixture thereof. The coupling
reaction can be carried out in N-methylpyrrolidone (NMP) or in DMF.

[0049] In another example, the coupling reaction can involve the treatment
of a sulfonamide with a protected hydroxylamine in the presence of EDC,
HOBt, and NMM in DMF. See Tamura et at., J Med Chem 1998,41,640-649,
which is incorporated by reference herein for its teaching of amine-acid
coupling reactions. Other conjugation techniques are disclosed in Greg T.
Hermanson, "Bioconjugate Techniques," Academic Press (Elsevier), 1996,
which is incorporated by reference herein for its teaching of conjugation
techniques. This embodiment of the invention can be combined with any of
the above disclosed compositions and methods.

[0050] Generally, the compositions of the invention can be administered in
a variety of forms, such as in a sterile aqueous solution or other
pharmaceutically acceptable carrier, among others. The aqueous solutions
can generally comprise a concentration of about 0.1 to 80 (e.g. 1-20)
percent by weight of the formulation or composition (e.g., the delivery
vehicle, retention vehicle, cross-linker, or combination thereof). In one
example, the formulation has a viscosity of at least about 10 cps, at a
shear rate of about 0.1/second. The present formulations are particular
useful as injectable formulations, which can be injected into a location
of a subject through a delivery device, such as a needle. The size of the
needle can be related to the size of the location that is being treated
and the formulation properties. In the case of human knee joints, for
example, the preferable needle size is about 18 gauge or smaller.

[0051] The formulations can be sterilized prior to use. For example, the
formulation can be sterilized by common sterilization methods, including
ionizing radiation, such as gamma radiation or electron beam radiation,
or ethylene oxide (EtO) exposure. Generally, the formulations can be
prepared by mixing the components manually or by other mechanical mixing
methods, subsequently loading the formulation into a delivery device,
such as a syringe, sterilized, and subsequently packaged, as an example
method for preparing the formulation.

[0052] The mode of administration can be any suitable mode, for example
subcutaneous injection, parental administration, enternal administration,
and the like. The formulations are preferably injected or simply placed
into a subject at a local site. Other conventional delivery modalities
include catheters, infusion pumps, pen devices and the like, all of which
can be used for local delivery of the formulation.

[0053] The compositions generally comprise an "effective amount" of the
bioactive agent, which refers to an amount of the formulation that will
achieve a desired therapeutic result. The effective amount will vary
greatly depending on the composition, bioactive agent, and disorder or
condition that is being treated. The actual effective amount of dosage
amount of the composition administered to a subject can be determined by
physical and physiological factors such as body weight, severity of
condition, the type of disease being treated, previous or concurrent
therapeutic interventions, idiopathy of the patient and can depend on the
route of administration. Depending upon the dosage and the route of
administration, the number of administrations of a preferred dosage
and/or an effective amount may vary according to the response of the
subject. One of skill in the art can determine an effective amount of a
disclosed pharmaceutical composition.

[0054] The therapeutically effective amount of a formulation for injected
into an affected area or treatment site is dependent on several factors,
including but not limited to location of the site and the size of the
desired area of injection. For example, a therapeutic amount of up to 5
mL of the formulation can be injected or infused into the human
intra-articular space of the knee. Formulation volume can be easily
adjusted by one of ordinary skill in this art for injections or delivery
into other areas, including joints, such as the hip, shoulders, ankles,
elbows, wrists, toes, fingers, and spinal facet joints. Up to 10 mL can
be injected or infused into the human subarachnoid space.

[0055] In some non-limiting examples, a dose of a bioactive agent which is
present within a composition of the invention can comprise from about 1
microgram/kg/body weight, about 5 microgram/kg/body weight, about 10
microgram/kg/body weight, about 50 microgram/kg/body weight, about 100
microgram/kg/body weight, about 200 microgram/kg/body weight, about 350
microgram/kg/body weight, about 500 microgram/kg/body weight, about 1
milligram/kg/body weight, about 5 milligram/kg/body weight, about 10
milligram/kg/body weight, about 50 milligram/kg/body weight, about 100
milligram/kg/body weight, about 200 milligram/kg/body weight, about 350
milligram/kg/body weight, about 500 milligram/kg/body weight, to about
1000 mg/kg/body weight or more per administration, and any range
derivable therein. In non-limiting examples of a derivable range from the
numbers listed herein, a range of about 5 mg/kg/body weight to about 100
mg/kg/body weight, about 5 microgram/kg/body weight to about 500
milligram/kg/body weight, etc., can be administered, based on the numbers
described above.

[0056] The formulation of the invention can also conveniently contain
epinephrine, dexamethasone, or other anti-inflammatory drugs which reduce
blood flow at the site of formulation infusion.

[0057] The compositions can be administered to any desired subject. The
subject can be a vertebrate, such as a mammal, a fish, a bird, a reptile,
or an amphibian. The subject of the herein disclosed methods can be, for
example, a human, non-human primate, horse, pig, rabbit, dog, sheep,
goat, cow, cat, guinea pig or rodent. The term does not denote a
particular age or sex. Thus, adult and newborn subjects, as well as
fetuses, whether male or female, are intended to be covered.

[0061] Other bioactive agents include but are not limited to analgesics
such as acetaminophen, acetylsalicylic acid, and the like; anesthetics
such as lidocaine, xylocaine, and the like; anorexics such as dexadrine,
phendimetrazine tartrate, and the like; antiarthritics such as
methylprednisolone, ibuprofen, and the like; antiasthmatics such as
terbutaline sulfate, theophylline, ephedrine, and the like; antibiotics
such as sulfisoxazole, penicillin G, ampicillin, cephalosporins,
amikacin, gentamicin, tetracyclines, chloramphenicol, erythromycin,
clindamycin, isoniazid, rifampin, and the like; antifungals such as
amphotericin B, nystatin, ketoconazole, and the like; antivirals such as
acyclovir, amantadine, and the like; anticancer agents such as
cyclophosphamide, methotrexate, etretinate, and the like; anticoagulants
such as heparin, warfarin, and the like; anticonvulsants such as
phenytoin sodium, diazepam, and the like; antidepressants such as
isocarboxazid, amoxapine, and the like;antihistamines such as
diphenhydramine HCl, chlorpheniramine maleate, and the like; hormones
such as insulin, progestins, estrogens, corticoids, glucocorticoids,
androgens, and the like; tranquilizers such as thorazine, diazepam,
chlorpromazine HCl, reserpine, chlordiazepoxide HCl, and the like;
antispasmodics such as belladonna alkaloids, dicyclomine hydrochloride,
and the like; vitamins and minerals such as essential amino acids,
calcium, iron, potassium, zinc, vitamin B12, and the like;
cardiovascular agents such as prazosin HCl, nitroglycerin, propranolol
HCl, hydralazine HCl, pancrelipase, succinic acid dehydrogenase, and the
like; peptides and proteins such as LHRH, somatostatin, calcitonin,
growth hormone, glucagon-like peptides, growth releasing factor,
angiotensin, FSH, EGF, bone morphogenic protein (BMP), erythopoeitin
(EPO), interferon, interleukin, collagen, fibrinogen, insulin, Factor
VIII, Factor IX, Enbrel®, Rituxan®, Herceptin®,
alpha-glucosidase, Cerazyme/Ceredose®, vasopressin, ACTH, human serum
albumin, gamma globulin, structural proteins, blood product proteins,
complex proteins, enzymes, antibodies, monoclonal antibodies, and the
like; prostaglandins; nucleic acids; carbohydrates; fats; narcotics such
as morphine, codeine, and the like, psychotherapeutics; anti-malarials,
L-dopa, diuretics such as furosemide, spironolactone, and the like;
antiulcer drugs such as rantidine HCl, cimetidine HCl, and the like.

[0064] Various modifications and variations can be made to the compounds,
composites, kits, articles, devices, compositions, and methods described
herein. Other aspects of the compounds, composites, kits, articles,
devices, compositions, and methods described herein will be apparent from
consideration of the specification and practice of the compounds,
composites, kits, articles, devices, compositions, and methods disclosed
herein. It is intended that the specification and examples be considered
as exemplary.